Molecular Medicine

Effect of the oxLDL Binding Protein Fc-CD68 on Plaque Extension and Vulnerability in AtherosclerosisNovelty and Significance

Stephan Zeibig, Zhongmin Li, Silvia Wagner, Hans-Peter Holthoff, Martin Ungerer, Andreas Bültmann, Kerstin Uhland, Jasmin Vogelmann, Thomas Simmet, Meinrad Gawaz, Götz Münch
https://doi.org/10.1161/CIRCRESAHA.111.240515
Circulation Research. 2011;108:695-703
Originally published March 17, 2011

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Abstract

Rationale: There is strong evidence that oxidative modification of low-density lipoprotein (oxLDL) plays a critical role in atherogenesis and that oxLDL may profoundly influence the mechanical stability of atherosclerotic plaques.

Objective: To block oxLDL, we designed, expressed, and tested Fc-CD68, a soluble oxLDL binding protein consisting of human Fc and the extracellular domain of the human oxLDL-binding receptor CD68.

Methods and Results: Fc-CD68 bound with high specific affinity to oxLDL and strongly bound and colocalized with oxLDL in plaques. To study the effects of repeated administrations of Fc-CD68 on the progression of atherosclerosis and plaque vulnerability, 12- and 16-week old cholesterol-fed ApoE−/− mice received either Fc-CD68 (n=6) or Fc control protein (n=6 to 8) thrice weekly for 4 weeks. Macroscopic and histological analysis of Sudan red lipid staining showed strong and significant reduction of plaque extension in the aorta and in the aortic root, respectively. Histological analysis of pentachrome- and Sirius-stained sections of the brachiocephalic arteries of 20 week-old ApoE−/− mice revealed that Fc-CD68 significantly reduced the occurrence of spontaneous ruptures of established plaques by ≈20%, compared with Fc and drastically increased the collagen content of plaques. Furthermore, in immunostained sections of the brachiocephalic artery and the aortic root, Fc-CD68 reduced the infiltration of plaques with T lymphocytes, and macrophages by ≈50% and 30%, respectively.

Conclusions: The oxLDL binding protein Fc-CD68 attenuates atherosclerosis and strengthens the stability of atherosclerotic plaques.

Oxidatively modified low-density lipoprotein (oxLDL) plays a critical role in atherogenesis and contributes to the progression of atherosclerosis in various ways.1,2 It can induce the transformation of macrophages into lipid-laden foam cells,3,4 it is a chemotactic agent for monocytes, and it reduces the motility of macrophages which then become resident in the arterial intima.5

In addition, oxLDL is recognized as foreign by the immune system.6 oxLDL-specific T lymphocytes are present in the vessel wall where they are locally restimulated and further stimulate macrophages by cytokine release.7,8 Furthermore, oxLDL is cytotoxic and damages the endothelium,9 thereby favoring platelet adhesion.10 In advanced atherosclerotic lesions, the cytotoxicity of oxLDL may even result in irreversible cell necrosis.10,11

In addition to contributing to atherosclerosis, oxLDL may profoundly influence the mechanical stability of atherosclerotic plaques, because foam cells offer little mechanical stability and because activated macrophages may secrete factors that weaken plaques, ie, metalloproteinases.12,13 Rupture of atherosclerotic plaques leads to vascular injury and subsequent myocardial infarction and stroke. Several studies have demonstrated conclusively that the morphology of the atherosclerotic plaques, rather than their size, is predictive for the frequency of plaque ruptures (summarized by Rekhter14). The most relevant morphological characteristics that determine the propensity of plaques to cause clinical events were found to be the relative thickness of the fibrous cap, consisting of collagen fibers and smooth muscle cells, the size of the lipid core, as well as the cellularity of the plaque, ie, the number of inflammatory cells such as macrophages and T lymphocytes within the intima. Therefore, a pharmacological agent that is able to stabilize plaques by influencing the above-named factors would be of great therapeutic benefit.

The oxLDL binding receptor CD68 and its murine homolog, macrosialin, are 94- to 97-kDa heavily glycosylated type I transmembrane proteins that are predominantly expressed on the cell surface of macrophages.15,16 Here, they might contribute among other oxLDL scavenger receptors, such as CD36, Lox-1, SR-A, and SR-B,17 to the specific uptake of oxLDL.18

A soluble recombinant dimeric fusion protein CD68-Fc consisting of the functional extracellular domain of CD68, a linker sequence and a Fc region of human immunoglobulin has recently been designed. The fusion protein had the capacity to bind oxLDL and lipid-rich structures of human atherosclerotic plaque specimen. Furthermore, CD68-Fc reduced the uptake of modified oxLDL by macrophages and platelets and the formation of foam cells, as well as the secretion of MMP-9, respectively.19 The fusion protein is thought to exert its atheroprotective effects by blocking its ligand oxLDL, which is assumed to interfere with oxLDL–macrophage interactions and to reduce the recognition of oxLDL by the immune system.

In this study, we further investigated a Fc-CD68 fusion protein in vivo for its atheroprotective and plaque stabilizing effect in ApoE−/− mouse models at different stages of atheroprogression.

Methods

An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.

Cloning, Expression, and Purification of Fc-CD68 and Fc

A Fc-CD68 fusion protein consisting of the leader sequence of IgGκ (IgK), the fragment crystallizable region of human IgG2 (Fc) with the hinge region, and the extracellular domain of the human CD68 receptor and a corresponding IgG2 Fc control protein without the extracellular domain of CD68 were designed (Figure 1A), cloned, expressed in Flp-In CHO expression cell lines (Invitrogen GmbH, Karlsruhe, Germany), and purified via Protein G affinity chromatography (Figure 1B).

Figure 1.
Figure 1.

Illustration of the soluble Fc-CD68 fusion protein. A, Schematic illustration of the fusion protein. B, Characterization of the fusion protein. Coomassie stained 4% to 20% polyacrylamide gradient gel, loaded under reducing conditions with molecular mass standard (lane 1), 17 μg of purified Fc (lane 2), and 20 μg of purified Fc-CD68 (lane 3), respectively. MW indicates molecular mass in kilodaltons. Arrows point to purified Fc and Fc-CD68.

Animals

Male homozygous apolipoprotein E knockout mice B6.129P2-ApoetmUnc (ApoE−/−) from in-house breeding were used. The housing and care of the animals and all procedures used in these studies were performed in accordance with the guidelines and regulations of the German Animal Welfare Act. The animal studies were approved by the local ethics committee with reference number 55.2-1-54-2531-141-07. Wild-type black six mice were used as controls.

Husbandry

The mice were kept according to standard housing conditions, except that ApoE−/− mice were fed with a modified chow based Paigen diet with 7.5% cocoa butter and 0.2% Na-cholate, containing 0.25% cholesterol (SSNIFF Spezialdiäten GmbH). Feeding with this cholesterol-rich diet was started at the age of four weeks and was continued throughout the experiment.

Study Design

For colocalization experiments, 20-week-old ApoE−/− mice (n=6) and wild-type mice (n=6) were injected once with 10 μg/g body weight Fc-CD68 or 3 μg/g Fc control and euthanized after 2 hours. Two treatment schedules were used for ApoE−/− mice. In one study, animals started to receive Fc-CD68 or Fc (n=6, each) at 16 weeks of age (model of spontaneous plaque rupture). In the other study, treatment with Fc-CD68 (n=6) and Fc (n=8) was started at 12 weeks of age (model of atheroprogression). In the models of spontaneous plaque rupture and in the model of atheroprogression, euthanasia was at the age of 20 weeks or 17 weeks, respectively (see Online Figure I).

Macroscopic Analysis of Plaque Extension

For macroscopic analysis of plaque extension, the adventitia was removed from the vessels and fixed overnight in paraformaldehyde. The aortas were incised, stained with Sudan III, mounted, and then photographed. Plaque areas and whole vessel area were determined and the relative plaque extension was expressed as percentage of the total plaque area of the whole vessel area.

Histology and Immunocytochemistry

Serial sections of 5 μm were cut every 75 μm along the brachiocephalic artery (from 20-week-old mice only) and every 80 μm along the 230-μm segment of the aortic root. The first tissue section of every segment was stained with hematoxylin/eosin. Adjacent sections were stained with oil red O, Sirius red, Movat's pentachrome,20 and van Gieson (elastin) and immunostained for the visualization of T lymphocytes, macrophages, the Fc part of Fc-CD68, or Fc and oxLDL as required.

Data Presentation and Statistics

Comparisons between group means were performed using Student t test where applicable. Data are presented as means±SEM. P≤0.05 was considered as statistically significant, P≤0.005 as highly significant.

Results

Design, Production, and Characterization of a Soluble oxLDL Binding Protein

Based on a recently characterized soluble oxLDL binding protein CD68-Fc,19 we designed and cloned Fc-CD68, a soluble recombinant fusion protein consisting of the crystallizable fragment of human IgG2 together with the hinge region and the functional extracellular domain of CD68 and an eligible IgG2-Fc control protein without the CD68 part (Figure 1A). Stable Flp-In CHO expression cell lines were generated for eukaryotic expression of Fc-CD68 and Fc. The fusion protein could be purified in one step from the supernatants of the suspension cultures to >95% purity. The apparent molecular mass deduced from SDS-PAGE of ≈110 kDa was considerably larger than the calculated molecular weight of the amino acid chain (56.6 kDa), indicating strong glycosylation (Figure 1B). SDS-PAGE analysis under reducing and nonreducing conditions confirmed the dimeric state of the fusion protein.

Fc-CD68 Binds to oxLDL With High Affinity

Surface plasmon resonance analysis revealed specific high affinity binding to modified LDL under reducing conditions in the presence of 1.43 mmol/L mercapto-ethanol with 10.6±2.3 nmol/L, whereas the binding of Fc-CD68 to native LDL was unspecific. Without reducing conditions the affinity of Fc-CD68 was 82.6±4.6 nmol/L to oxLDL and 67.0±11 nmol/L to LDL. No binding to HDL was detected.

Fc-CD68 and oxLDL Localization in ApoE−/− Mice and Wild-Type Mice

Localization of both Fc-CD68 and oxLDL could be detected in plaques of 20-week-old, cholesterol-fed ApoE−/− mice after single injection of Fc-CD68. For better comparability, matching immunostaining of Fc-CD68 (by detection of human Fc) and of oxLDL are shown in the same histological slice. Lipid extension and foam cell distribution is shown by sudan oil red staining in comparison (see Figure 2A). In contrast, in ApoE−/− mice injected with Fc control protein, no signal for Fc was detected, whereas oxLDL expression matching with the lipid distribution in the plaques is shown (see Figure 2B). The healthy wild-type mice treated with Fc-CD68 showed neither Fc-CD68 signal nor oxLDL immunofluorescence was seen (see Figure 2C).

Figure 2.
Figure 2.

Colocalization of Fc-CD68 and oxLDL. Colocalization of Fc-CD68 and oxLDL is demonstrated in 20-week-old cholesterol-fed ApoE−/− mice injected once intravenously with 10 μg/g Fc-CD68. Controls of ApoE−/− mice with 3 μg/g Fc injections or wild-type mice were used for comparison. In histological sections of the aortic arch (20-fold magnification), localization of oxLDL and strong Fc-CD68 colocalization is demonstrated by immunohistochemistry (A). Lipid distribution and foam cell formation in atherosclerotic plaques is visualized by oil red O staining. After single Fc-control injection, only oxLDL-positive immunohistochemistry is apparent and the absence of Fc binding (B). In contrast, in wild-type mice after Fc-CD68 injection, no oxLDL immunohistochemistry and the absence of Fc-CD68 binding together with no signs of atherosclerosis are visible in standard oil red staining (C).

Fc-CD68 Treatment Was Tolerated Well by All Mice

We assessed the atheroprotective effect of Fc-CD68 in 2 ApoE−/− mouse models of atherosclerosis. In a classical model, referred to as “atheroprogression model,” the prevention of atherogenesis by Fc-CD68 was studied using IP administrations of either Fc-CD68 (n=6) or Fc control protein (n=8) thrice weekly for four weeks starting from week 12. In the other, so-called “rupture model,” the same treatment regimen was applied for 4 weeks but treatment was started at 16 weeks of age (n=6 for each group). According to pilot experiments, at that time, atherosclerotic lesions already have been established, and plaques in the brachiocephalic artery were prone to spontaneous rupture. In both ApoE−/− mouse models, Fc-CD68 treatment was tolerated well by all mice. No unusual clinical abnormalities were observed during or after injection of Fc-CD68 or Fc, respectively, and no abnormal findings were observed on necropsy. The mean body weights did not differ significantly between Fc-CD68–treated animals and control animals at beginning or during the investigation period. Likewise, no significant difference was found in the mean weights of hearts, liver, spleen, and kidney after perfusion fixation in situ.

Fc-CD68 Attenuated Atheroprogression in the Aortas of Atherosclerotic ApoE−/− Mice

The effect of Fc-CD68 on plaque formation in the aorta was determined macroscopically on the basis of Sudan III–stained thoracic and abdominal aorta segments of mice of both models (Figure 3A). In the 20 week-old ApoE−/− mice of the rupture model, the plaques covered in average 32.5±3.7% and 18.9±2.6% of the thoracic and abdominal aorta in the Fc control group and 28.3±3.3% and 9.5±3.0% in the Fc-CD68 treatment group, respectively (Figure 3B). Accordingly, whereas the fusion protein only slightly reduced plaque extension in the thoracic aorta (P=NS), plaque extension was strongly and significantly reduced in the abdominal aorta by approx 50% (P=0.04).

Figure 3.
Figure 3.

Effect of Fc-CD68 on the plaque extension in the aorta. A, Representative photographs of Sudan III–stained thoracic and abdominal aortas. Scale bar, 1 cm. B, Quantitative analysis of the plaque extension. The mean percentages of plaque areas of total vessel areas of all mice of 1 group are shown with SEM (n=6 and 8 for the Fc and Fc-CD68 groups of the rupture model, respectively, and n=6 for both groups of the atheroprogression model). *P≤0.05, statistical significance; **P≤0.005, high statistical significance.

In the 17-week-old ApoE−/− mice of the atheroprogression model, the effects of Fc-CD68 on plaque extension were even more pronounced. Here the plaque covered on average 20±2.3% and 10.6±3.3% of the thoracic and abdominal aorta in the Fc control group and only 7.8±1.1% and 2.7±0.8% of the Fc-CD68 treatment group, respectively (Figure 3B). Thus in this study, plaque extension was reduced by 61% (P=0.0002) and 74% (P=0.01) in the thoracic and abdominal aorta, respectively.

Fc-CD68 Reduced Lipid Deposition in the Aortic Root of Atherosclerotic ApoE−/− Mice

The effect of Fc-CD68 on plaque extension in the aortic origin was evaluated by analyzing lipid deposition on oil red O–stained tissue sections of the aortic root (Figure 4A). In mice of the rupture model, the mean areas of lipid deposition in the aortic root were 0.015±0.003 mm2 and 0.045±0.004 mm2 for mice of the Fc-CD68 and Fc group, respectively (Figure 4B), corresponding to a reduction of lipid disposition to one third (P=0.0001). Likewise, in mice of the atheroprogression model, Fc-CD68 also had the capacity to reduce lipid deposition to one third (P=0.0005).

Figure 4.
Figure 4.

Effect of Fc-CD68 on lipid deposition in the aortic root. A, Representative photographs of oil red O–stained tissue sections of the aortic root. Scale bar, 400 μm. B, Quantification of the effect of Fc-CD68 on lipid deposition. The mean areas of all mice of the Fc-CD68 groups (n=7 and n=6 for the model of plaque rupture and atheroprogression, respectively) were normalized by referring to the mean areas of the respective Fc control groups (n=6 for each model). Means are shown with SEM. **P≤0.005, high statistical significance.

In both studies, the mean length of the external elastic lamina of the Fc-CD68 and Fc groups, evaluated on adjacent van Gieson stained sections, was nearly identical, allowing to exclude bias effects of cutting position (data not shown).

Systemic levels of HDL, LDL, and triglycerides were not significantly affected (Table). In both the atheroprogression (1.19±0.52), and in the plaque rupture model ApoE−/− mice (2.43±0.95), no significant anti Fc-CD68 antibody titers were detected compared with age-matched controls. IL-6 levels were unaffected and IL-8 levels slightly increased with Fc-CD68 (IL-6: control group: 0±0 pg/mL; Fc-CD68–treated group: 0±0 pg/mL; IL-8: control group: 60.7±93.4 pg/mL; Fc-CD68–treated group: 534.0±29.1 pg/mL). However, control Fc fusion protein also increased IL-8 levels (IL-8 Fc treated group: 258.4±134.4 pg/mL). Complement 5 (C5) levels remained largely unaffected by Fc-CD68 (control group: 35.4±6.5 ng/mL; Fc-CD68–treated group: 51.5±10.0; Fc group: 49.6±7.9 ng/mL).

View this table:
Table.

Effect of Fc-CD68 on Plasma Lipid Levels

Fc-CD68 Stabilized Plaques in the Brachiocephalic Artery of Atherosclerotic ApoE−/− Mice

For each animal of the rupture model, the effect of the Fc-CD68 on the frequency of spontaneous plaque ruptures was assessed (Figure 5A) on 12 Pentachrome stained tissue sections of the brachiocephalic artery in accordance with reference 21. Treatment with Fc-CD68 reduced the mean numbers of acute ruptures per section from 0.34±0.1 to 0.18±0.1, ie, by ≈50% compared with the control group. Because of the low frequencies of acute rupture events per section, significance could not be reached. The mean numbers of buried fibrous caps per section were significantly reduced by >20% from 1.89±0.05 in the Fc control group to 1.56±0.09 in the Fc-CD68 treatment group (P≤0.017). Taken together, the total number of rupture events, ie, acute ruptures plus buried caps, was significantly reduced by ≈20% (P=0.04) (Figure 5B).

Figure 5.
Figure 5.

Effect of Fc-CD68 on plaque ruptures in the brachiocephalic artery. A, Representative photographs of pentachrome- and hematoxylin/eosin (HE)-stained sections of the brachiocephalic artery of mice of the rupture model showing the typical appearance of thrombi, hemorrhage, and buried fibrous caps, respectively. B, Quantification of rupture events. The occurrences of acute ruptures and buried caps were counted on 12 cross-sections per mouse and used to average the mean occurrences per cross section. Means of all mice of one group (n=6) are shown with SEM. *P≤0.05, statistical significance.

Fc-CD68 Increased the Collagen Content of Plaques in Atherosclerotic ApoE−/− Mice

Whereas lipid deposits make plaques more prone to rupture, collagen fibers (mainly the thickness of the fibrous cap) stabilize plaques.14 Therefore, we assessed the effect of Fc-CD68 on the composition of plaques in the brachiocephalic artery. The areas of lipid deposits and the areas of collagen fibers within a plaque were determined on oil red O and adjacent Sirius stained tissue sections, respectively, (Figure 6A) and the ratio was calculated. Whereas the mean areas of lipid deposition of the plaques only showed a slight tendency to decrease in the Fc-CD68–treated mice (14.8±4.2 mm2), compared with control mice (17.2± 4.0 mm2), the mean collagen content of Fc-CD68–treated mice was increased by 77% from 40.6±3.3 mm2 to 71.8±4.1 mm2 (P≤0.005) (Figure 6B). Consequently, the collagen/lipid ratio of Fc-CD68–treated mice was more than twice as high as in control mice (9.3 versus 4.0), supporting the observation that the fusion protein has a plaque stabilizing effect (Figure 6C).

Figure 6.
Figure 6.

Effect of Fc-CD68 on the composition of plaques in the brachiocephalic artery. A, Representative photographs of Sirius red–stained (collagen) and oil red O–stained (lipids) sections of the brachiocephalic artery of atherosclerotic ApoE−/− mice of the rupture model. Scale bar, 200 μm. B, Quantification of lipid deposition and collagen content. Mean areas of lipid deposition and collagen fibers of all mice of one group (n=6) are shown with SEM. C, Plaque stability was assessed by forming the collagen to lipid ratio.

Fc-CD68 Reduced the Infiltration of Plaques With Inflammatory Cells and oxLDL Accumulation in Atherosclerotic ApoE−/− Mice

Because in addition to the plaque composition, inflammation is thought to have a great impact on the vulnerability of plaques,22 we assessed the degree of inflammation of plaques in Fc-CD68 and Fc treated mice of the rupture model. To do so, tissue sections of the aortic origin were immunostained with specific markers for macrophages (F4/80) and T lymphocytes (CD3), respectively (Figure 7A).

Figure 7.
Figure 7.

Effect of Fc-CD68 on the infiltration of plaques with inflammatory cells. A, Representative photographs of sections of the aortic root of mice of the rupture model that were immunostained with either the T-lymphocyte–specific marker CD3 or the macrophage specific marker F4/80. Scale bars, 100 μm. B, Quantification of the infiltration of plaques with inflammatory cells. The relative densities of T lymphocytes and macrophages, respectively, were obtained by dividing the numbers of specifically stained immune cells by the associated intima area. C, Localization of oxLDL in 20-week-old ApoE−/− mice. Representative photographs of sections of the brachiocephalic artery of mice of the rupture model that were immunostained with by immunocytochemistry with specific anti-malondialdehyde antibodies. Scale bars, 100 μm. Quantification of the staining of plaques by densitometric analysis of digitalized images is shown. Mean ratios of all mice of 1 group (n=6) are shown with SEM. *P≤0.05.

The mean relative densities of T lymphocytes and macrophages, ie, the numbers of CD3+ and F4/80+ cells per analyzed area, were 57.3±6.8 nuclei/mm2 and 233.6±28 nuclei/mm2 for mice of the Fc-CD68 group and 121.9±25.9 nuclei/mm2 and 322.8±29.1 nuclei/mm2 for the Fc group, respectively (Figure 7B). Hence, Fc-CD68 significantly reduced the mean relative infiltration of plaques with T lymphocytes and macrophage in the aortic root by 53.0% (P=0.04) and 27.6% (P=0.05), respectively. Comparable results were obtained in the brachiocephalic artery. oxLDL was slightly and significantly decreased from 14.3±0.4 densitometric units to 12.7±0.4 in the aortic root of 20-week-old Fc-CD68–treated ApoE −/− mice and from 14.7±0.5 densitometric units to 12.8±0.6 in 16-week-old Fc-CD68–treated ApoE −/− mice.

Plasma CRP levels of Fc-CD68 and Fc treated mice were 45.8±6.0 and 51.3±14.3 μg/mL, respectively, in the rupture model and 14.2±7.6 and 19.4±17.4 μg/mL, respectively, in the atheroprogression model. In both models, there was no significant difference between treatment and control group. The fusion protein thus reduced the local inflammation at atherosclerotic plaques but did not affect systemic inflammation.

Discussion

We directly assessed the effect of Fc-CD68 on spontaneous plaque rupture in the brachiocephalic artery, the only location prone to spontaneous plaque rupture in ApoE−/− mice.23 Fc-CD68 achieved a 50% reduction of acute ruptures and 20% reductions of buried caps and total rupture events, respectively, compared with the Fc control protein. For the reduction of clinical events with potential fatal consequence, this reduction is quite high and having in mind that in our rupture model treatment was only started at 16 weeks of age, when some rupture events might very likely already have occurred, the efficacy of the fusion protein to prevent rupture events might even be underestimated. The plaque-stabilizing effect of the Fc-CD68 fusion protein was strongly supported by the assessment of indirect indicators of plaque vulnerability. The infiltration of lesions with T lymphocytes and macrophages was reduced by ≈50% and 30%, respectively, and, most likely as a direct consequence of the reduced number and activation of matrix metallo proteinases (collagenases)- secreting macrophages,19,24,25 the collagen content was ≈1.8-fold higher in Fc-CD68–treated mice than in Fc treated mice. Fc-CD68 also slightly but significantly reduced oxLDL accumulation in atherosclerotic plaques. In contrast to local inflammation, the systemic inflammatory biomarker CRP was not suppressed. Moreover, other systemic inflammatory markers such as IL6 and IL8 or complement C5 were also not affected by Fc-CD68. This observation is not surprising, when bearing in mind that the mice were persistently fed with a high fat cholate containing diet, the proinflammatory nature of which might easily conceal any antiinflammatory effects.

Simultaneously, Fc-CD68 was capable to decelerate atheroprogression. It strongly reduced lipid deposition and plaque extension in the aortic root and aorta, respectively. As reported earlier,26 in our model the progression of atherosclerosis was faster in the aortic arch than in the descending part of the thoracic aorta and faster in the thoracic aorta than in the abdominal aorta. Our observation that in the 20-week old mice the fusion protein strongly and significantly reduced the plaque extension in the abdominal aorta but only slightly in thoracic aorta indicates that Fc-CD68 prevents more efficiently atheroprogression in earlier lesions than in advanced lesions. This hypothesis is further supported by our finding that the effect on plaque progression was more pronounced in younger mice (atheroprogression model) than in older mice (rupture model).

We selected the isotype control to assess the specific effects of the CD68 part of our fusion protein. Because it was shown recently in a similar experimental setting, that macrophage and lipid content of plaques did not differ significantly in Fc isotype control and vehicle control (PBS) mice,27 the effect of Fc treatment can be neglected in this model. According to these published data, there is sufficient evidence that treatment effects with Fc-CD68 would remain about the same, if compared with vehicle instead of Fc. Using the isotype controls, the observed effects can most likely be attributed specifically to the CD68 part of the fusion protein.

Oxidized LDL is recognized by the immune system28,29 and studies in rabbits and mice have suggested that specific adaptive immune responses exert protective effects against atherosclerosis.30,–,32 Pertinent to antigen induced anti-oxLDL antibodies, recombinant anti-oxLDL antibodies were recently shown to reduce the progression of atherosclerosis and plaque inflammation in ApoE−/− and Apobec-1−/−/low density lipoprotein receptor−/− mice, respectively.33,34 Fc-CD68 mimics such anti-oxLDL antibodies in that it consists of two oxLDL binding domains, ie, extracellular domains of human CD68 that are linked via disulfide bond in the Fc part. In intravenous immunoglobulin preparations (IVIGs) anti-idiotypic “anti–anti-oxLDL antibodies” were found and neutralized 65% to 90% of the oxLDL binding capacity of anti-oxLDL antibodies.35 As no anti-Fc-CD68 were detected in this study, we do not expect Fc-CD68 to induce neutralizing anti-CD68 antibodies in humans, so that in contrast to anti-oxLDL antibodies, Fc-CD68 should retain its full binding activity even after repeated administrations.

Our concept to fuse the oxLDL binding regions of receptors to Fc has recently been followed by two other groups. In one approach, the extracellular domain of the oxLDL scavenger receptor CD36 was fused to the Fc domain of human IgG1 and the resulting chimeric sCD36-Ig was able to inhibit the adhesion of monocytes to oxLDL.36 Likewise, recombinant Lox1-Fc fusion proteins were shown to inhibit binding and internalization of oxLDL by cell surface Lox-1, but only when the construct was oligomerized via a polyclonal antibody against Fc.37 These approaches support the in vitro results of CD68-Fc19 and point to one mode of action of soluble Fc fusion proteins, ie, impeding the binding of oxLDL to scavenger receptors and interfering with oxLDL uptake by monocytes and associated foam cell formation. Moreover, in colocalization experiments, Fc-CD68 and oxLDL was found in similar regions of the plaques of ApoE−/− mice with concentration in the lipid rich parts or foam cell rich parts of the plaques. These activities indicate that Fc-CD68 might exert its effects by binding to oxLDL in the plaque and reducing the accumulation of oxLDL in foam cells as previously shown in vitro together with reducing its proinflammatory effects.19 On the other hand, reduced binding of oxLDL at the cell surface might reduce the number of macrophages attracted by the chemotactic agent oxLDL. In addition, binding of Fc-CD68 to oxLDL might “coat” and hence conceal oxLDL from the immune system.

Anti-oxLDL antibodies were not only reported to have beneficial effects but were also associated with cardiovascular disease.38 Nagarajan suggested that the interaction between Fcγ receptors expressed on inflammatory cells and oxLDL immune-complexes and associated cytokine release as one potential mechanism of how anti-oxLDL antibodies might contribute to the progression of atherosclerosis.39 To reduce such Fcγ receptor interactions, in our new construct, we exchanged the Fc region of the fusion protein from IgG1 to IgG2, the latter being suggested as the isotype of choice for “effector-function-silent” Fc fusion proteins.40

In conclusion, repeated systemic administration of Fc-CD68, a new oxLDL binding protein with favorable characteristics, resulted in a significant inhibition of the progression of atherosclerosis and promoted plaque stabilization in areas of the vasculature predisposed to plaque formation. Fc-CD68 might therefore offer a potent therapeutic strategy for the prevention and therapy of atherosclerosis.

Sources of Funding

The work was funded by the Gründungs-Offensive Biotechnologie (Go-Bio) initiated by the Bundesministerium für Bildung und Forschung (BMBF, German Ministry for Education and Research), grant 0315363 and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) grant Li 849/3-1 and SFB-TR 19 to Meinrad Gawaz).

Disclosures

None.

Acknowledgments

We acknowledge the excellent technical assistance of Ulrike Potschka and Melanie Schauber.

Footnotes

  • In December 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.5 days.

  • Non-standard Abbreviations and Acronyms

    Apo
    apolipoprotein
    Fc-CD68
    fusion protein of the crystallizable fragment of the immunoglobulin γ 2 and the extracellular domain of the human oxLDL binding receptor CD68
    oxLDL
    oxidative modification of low-density lipoprotein

  • Received February 17, 2010.
  • Revision received January 11, 2011.
  • Revision received January 22, 2011.
  • Accepted January 25, 2011.

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Novelty and Significance

What Is Known?

  • Oxidative modification of low-density lipoprotein (oxLDL) is believed to be an important trigger of atherosclerotic lesion development and progression.

  • Different receptors on macrophages can bind to oxLDL.

What New Information Does This Article Contribute?

  • Recombinant, soluble CD68 receptor (Fc-CD68) colocalizes with and binds to oxLDL in the plaques of atherosclerotic mice in vivo.

  • Fc-CD68 attenuates atheroprogression.

  • Fc-CD68 reduces the rupture of atherosclerotic plaques in a mouse model.

Accumulation of oxLDL in macrophages is considered the first step in atherogenesis leading to foam cell formation and initiating inflammation in atherosclerotic plaques. This inflammation destabilizes the plaques, with consequent plaque rupture and arterial thrombosis. In this study, we show that CD68, a surface receptor on macrophages and oxLDL binding receptor, is required for the accumulation of oxLDL in atherosclerotic plaques. In ApoE−/− mice, Fc-CD68 attenuated atheroprogression and reduced macrophage and T-cell accumulation and oxLDL content in the plaques. The plaque was stabilized with a relative reduction of the lipid core and an increase in the collagen content of the fibrous cap. Plaque rupture was significantly decreased by Fc-CD68 treatment in these ApoE−/− mice. Our findings support the novel concept that Fc-CD68 may be a useful therapeutic approach for preventing fatty streak formation and plaque rupture.

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  • Effect of the oxLDL Binding Protein Fc-CD68 on Plaque Extension and Vulnerability in AtherosclerosisNovelty and Significance
    Stephan Zeibig, Zhongmin Li, Silvia Wagner, Hans-Peter Holthoff, Martin Ungerer, Andreas Bültmann, Kerstin Uhland, Jasmin Vogelmann, Thomas Simmet, Meinrad Gawaz and Götz Münch
    Circulation Research. 2011;108:695-703, originally published March 17, 2011
    https://doi.org/10.1161/CIRCRESAHA.111.240515
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